The Molecular Effect of Wearing Silver-Threaded Clothing on the Human Skin

ABSTRACT With growing awareness that what we put in and on our bodies affects our health and wellbeing, little is still known about the impact of textiles on the human skin. Athletic wear often uses silver threading to improve hygiene, but little is known about its effect on the body’s largest organ. In this study, we investigated the impact of such clothing on the skin’s chemistry and microbiome. Samples were collected from different body sites of a dozen volunteers over the course of 12 weeks. The changes induced by the antibacterial clothing were specific for individuals, but more so defined by gender and body site. Unexpectedly, the microbial biomass on skin increased in the majority of the volunteers when wearing silver-threaded T-shirts. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. Both effects were mainly observed for women. The hallmark of the induced changes was an increase in the abundance of various monounsaturated fatty acids (MUFAs), especially in the upper back. Several microbe-metabolite associations were uncovered, including Cutibacterium, detected in the upper back area, which was correlated with the distribution of MUFAs, and Anaerococcus spp. found in the underarms, which were associated with a series of different bile acids. Overall, these findings point to a notable impact of the silver-threaded material on the skin microbiome and chemistry. We observed that relatively subtle changes in the microbiome result in pronounced shifts in molecular composition. IMPORTANCE The impact of silver-threaded material on human skin chemistry and microbiome is largely unknown. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. The major change was an increase in the abundance of various monounsaturated fatty acids that were also correlated with Cutibacterium. Additionally, Anaerococcus spp., found in the underarms, were associated with different bile acids in the armpit samples. Overall, the impact of the silver-threaded clothing was gender and body site specific.


Summary
This manuscript, entitled "The molecular effect of wearing silver-threaded clothing on the human skin", presents interesting and novel information that is of interest of mSystems and its audience. They investigated the impact of clothes with silver threading on the chemistry and microbiome of the human skin.
Silver-threaded clothing are often wore by athletes to improve hygiene. Counterintuitively, the authors observed an increase on microbial biomass on skin when wearing this kind of clothing. Even though the most abundant taxa were not affected, an increase in diversity and richness of low-abundant bacteria was observed. Chemical diversity was decreased, and an increase in the abundance of various monounsaturated fatty acids (MUFAs) was observed. Notably, results varied among volunteers and body sites, which major changes being mainly observed for the female group and upper back. This study contributes to the understanding of how textile influence our skin metabolome and microbiome, raising the awareness on the impact of our daily choices of clothing on skin health.
Overall, the manuscript is well-written. The authors planned well their experiments, used appropriated controls, and did a good and deep statistical analysis of their data. A limitation of this study is that a high variability among volunteers was observed, which is expected, and they acknowledged that further studies with a higher number of volunteers is needed to better understand this topic.
I have some minor concerns, shown below. I would like to suggest that next time authors add line numbers to the text to facilitate the review process.

Minor concerns
-As stated in the manuscript, there was variability among volunteers in the biological and chemical response to wearing clothes with silver threading. Nobody has identical microbiomes, and this natural variability from person to person might be in part responsible for the variability observed in the outcomes of this study. With this in mind, it would be interesting to add to the supplemental information a comparison of the microbiome at time zero among volunteers. How different are they? What are the major differences?
Maybe this information could be useful to understanding the variability in the outcomes after wearing silver threading.
-In the methods section it is disclosed that females wore clothes with a higher silver content (5% versus 4% in male clothes), and this information is not presented in any other section. The study showed that changes were more pronounced in the females group. Maybe a higher silver content could be one of the factors driving the observed difference. Authors should acknowledge this in the text.
-It is mentioned in the manuscript that silver threading is often wore by athletes to control odour and improve hygiene due to its antimicrobial property. However, the results show the oppositea higher bacterial biomass was observed, as well as a higher abundance of some odour-causing species, such as Anaerococcus. It would be interesting to add this perspective to the discussion, that the silver threading is not behaving as it is expectedit is actually doing the opposite. This highlights the importance of this kind of microbiome/metabolome study when developing a functional textile.
-The defined noise level (during mass spec data processing) seems very low (1000). Were the signals in general at low abundance? Why such a low noise level was chosen? -What is the confidence level of the annotated compounds? How were they annotated? Please add this information to the text.
-Page 6, last sentence of the second paragraph: where it says " Figure 1a", it is actually 1b, and viceversa.
-Page 7, Figure 1: please add to the figure legend the meaning of: size of the nodes, edge thickness, and numbers inside the nodes.
-Page 9, figure 2a and 2b: I'm not convinced by these plots that silver and non-silver groups are different in the upper and lower back. I don't see any separation of these groups in these 2 PCoA plots, which is confusing given that the text says "When considering body parts separately, the effects of silver could be more clearly observed". I would like to ask the authors to please elaborate more the discussion of these plots.
-Page 10, second last line: "Corynebacterium spp. Were found to be one of the species that tend to cooccur with". Typo: "Were" should be written in lowercase. -Page 16, "Study Design" topic, line 12: there is a typo: "Srudy". In the same paragraph, the period is missing at the end of the paragraph. - Supplementary Figure 4: There are some colors in the color-code chart that I didn't find in the heatmap, like lower back and blank. If they are indeed not present, please update the color-code accordingly.
However, I suggest that it is only kept in the figure samples that are from armpits, since the point of this figure is to showcase the chemical differences between right and left armpit: "A number of compounds that may be linked to bacterial origin -acylcarnitines, phospholipids and bile acids have been found to have different abundances in two armpits". The way this figure is presented makes it hard to see this point: there's no explanation about tree diagrams on top and right side of the figure, and it is not described how they were constructed. Please add this information to the figure legend and/or methods section. I can't understand this "Color key and Histogram". What is "value" and "count"? The "count" axis goes from 0-80000, but I don't see anything in the plot, I guess the histogram is missing in this plot? -Supplementary Figure 5A: add "(qPCR)" to the axis title: "16S copies per uL (qPCR)". Reviewer #1 (Comments for the Author): I would like to suggest that next time authors add line numbers to the text to facilitate the review process.
A: We appologize for the inconvenience regarding this issue. We would definitely consider this for the next submission Summary This manuscript, entitled "The molecular effect of wearing silver-threaded clothing on the human skin", presents interesting and novel information that is of interest of mSystems and its audience. They investigated the impact of clothes with silver threading on the chemistry and microbiome of the human skin. Silver-threaded clothing are often wore by athletes to improve hygiene. Counterintuitively, the authors observed an increase in microbial biomass on skin when wearing this kind of clothing. Even though the most abundant taxa were not affected, an increase in diversity and richness of low-abundant bacteria was observed. Chemical diversity was decreased, and an increase in the abundance of various monounsaturated fatty acids (MUFAs) was observed. Notably, results varied among volunteers and body sites, which major changes being mainly observed for the female group and upper back. This study contributes to the understanding of how textile influence our skin metabolome and microbiome, raising the awareness on the impact of our daily choices of clothing on skin health. Overall, the manuscript is well-written. The authors planned well their experiments, used appropriated controls, and did a good and deep statistical analysis of their data. A limitation of this study is that a high variability among volunteers was observed, which is expected, and they acknowledged that further studies with a higher number of volunteers is needed to better understand this topic. I have some minor concerns, shown below. I would like to suggest that next time authors add line numbers to the text to facilitate the review process.
Minor concerns -As stated in the manuscript, there was variability among volunteers in the biological and chemical response to wearing clothes with silver threading. Nobody has identical microbiomes, and this natural variability from person to person might be in part responsible for the variability observed in the outcomes of this study. With this in mind, it would be interesting to add to the supplemental information a comparison of the microbiome at time zero among volunteers. How different are they? What are the major differences? Maybe this information could be useful to understanding the variability in the outcomes after wearing silver threading.
A: Thank you. There is indeed a certain interindividual variability we need to take into account. We agree with the referee that it would be useful to add a graph with microbiome compositions on timepoint 0 in supplemental information. We have added this information as Supplemental Figure 10. It is also referred to in the main manuscript in the microbiome part of the Results section. It reads: "The high interindividual response in skin microbiome was further backed by the initial individual skin microbiome differences (Supplementary Figure  10)." -In the methods section it is disclosed that females wore clothes with a higher silver content (5% versus 4% in male clothes), and this information is not presented in any other section. The study showed that changes were more pronounced in the females group. Maybe a higher silver content could be one of the factors driving the observed difference. Authors should acknowledge this in the text. R: Thank you, I have added this point on the differences in silver content between males and female t-shirts in the results and discussion to read as follows: "This difference between males and females could have been due to the slightly higher content of silver in the females t-shirts. The amount of x-static silver threaded yarn was 5% for the female shirt and 4% for the male shirt." -It is mentioned in the manuscript that silver threading is often wore by athletes to control odour and improve hygiene due to its antimicrobial property. However, the results show the opposite -a higher bacterial biomass was observed, as well as a higher abundance of some odour-causing species, such as Anaerococcus. It would be interesting to add this perspective to the discussion, that the silver threading is not behaving as it is expected -it is actually doing the opposite. This highlights the importance of this kind of microbiome/metabolome study when developing a functional textile. A: Very good point. We have clarified these surprising counter-intuitive findings in the discussion section. The second paragraph of the discussion section now reads as follows: "Silver-coated yarn threaded T-shirts had a small but significant impact on the skin microbiome (mainly for females), leading to a higher richness and diversity including higher abundance of some odor-causing species, such as Anaerococcus. During the silver phase in male participants, a higher bacterial biomass was found with Staphylococcus and Corynebacterium being the most dominant ( Supplementary Figure 5d and 7). These results are counter-intuitive and surprising, since the primary reason for including silver-ions into garments is to manage or reduce the bacterial load and to control odor formation. This study shows the opposite outcome for the intended primary reason for including silver particles, as a higher bacterial load on skin was found, and particular malodor-associated taxa were increased on skin." The last paragraph of the discussion now reads: "In this paper the silver textile has caused slight distortion in the microbial community, which has led to these larger changes in metabolism on the skin. This highlights the importance of this kind of microbiome/metabolome study when developing a functional textile." (We couldn't have said it better) -The defined noise level (during mass spec data processing) seems very low (1000). Were the signals in general at low abundance? Why such a low noise level was chosen? A: During data acquisition we didn't want to omit any signal to construct reliable chromatograms. We did increase the threshold in feature finding to 3000 to filter out any noise.
-What is the confidence level of the annotated compounds? How were they annotated? Please add this information to the text. A: We have added a new paragraph to method section called "Metabolite annotation": MZmine preprocessed MS/MS fragmentation spectra were submitted to feature-based mass spectral molecular networking through the Global Natural Products Social Molecular Networking Platform (GNPS) [refs] and searched against all GNPS spectral libraries. The exact mass and MS/MS spectral matching is equivalent to the level 2 identification according to the paragraph 2.9 of "Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI)"(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772505/) To further enhance chemical structural information we performed in silico structure annotation using Network -Page 6, last sentence of the second paragraph: where it says " Figure 1a", it is actually 1b, and vice versa. A: Thank you for catching this, the last sentence of the second paragraph now reads: "T-shirt phase (Figure 1a) or different body parts (Figure 1b) gives visualizations of associated molecular distributions." -Page 7, Figure 1: please add to the figure legend the meaning of: size of the nodes, edge thickness, and numbers inside the nodes. A: We have added the discretion to the legend which now reads: Figure 1. Molecular networks for exploration of metabolome changes induced by silver fabric. Global molecular networks of metabolomics data color-coded by a) T-shirt phase and b) body part. c) The plot showing the ratio of abundances of metabolites during the silver versus non-silver phases on upper and lower back. Ratios higher and lower than one (i.e. molecules that increased and decreased in abundance) are highlighted in red and blue, respectively. Examples of some of the annotated molecules with the corresponding clusters from the molecular network that contain them are shown: monounsaturated fatty acids; bile acids and a surfactant. Clusters are colored the same way as in panel a). Numbers inside cluster nodes denote m/z as measured by a mass spectrometer, while size of the nodes is determined by the peak area under the curve. Thickness of the edges connecting nodes increases continuously with cosine similarity score increase. Consistent ratios of MUFAs' abundances across the cluster are suggestive of the same chemical forces responsible for the changes in their differences in silver vs. non-silver samples. The depicted compounds are highlighted by square nodes.
-Page 9, figure 2a and 2b: I'm not convinced by these plots that silver and non-silver groups are different in the upper and lower back. I don't see any separation of these groups in these 2 PCoA plots, which is confusing given that the text says "When considering body parts separately, the effects of silver could be more clearly observed". I would like to ask the authors to please elaborate more the discussion of these plots.
A: We have clarified the statement to read as follows: When considering body parts separately in unsupervised analysis, the effects of silver cannot be clearly observed: Figure 2a and 2b shows PCoA plots for the upper and lower back samples with some separation of samples based on silver and non-silver groups. Figure 2c shows the volcano plot for these samples. Several features appeared to be significant in discriminating silver and non-silver sample groups The features were predominantly attributed to a single network cluster shown on Figure 1c, indicating their structural similarity -Page 10, second last line: "Corynebacterium spp. Were found to be one of the species that tend to co-occur with". Typo: "Were" should be written in lowercase. A: Thank you for catching this typo. Adjusted. - Supplementary Figure 4: There are some colors in the color-code chart that I didn't find in the heatmap, like lower back and blank. If they are indeed not present, please update the color-code accordingly. However, I suggest that it is only kept in the figure samples that are from armpits, since the point of this figure is to showcase the chemical differences between right and left armpit: "A number of compounds that may be linked to bacterial originacylcarnitines, phospholipids and bile acids have been found to have different abundances in two armpits". The way this figure is presented makes it hard to see this point: there's no explanation about tree diagrams on top and right side of the figure, and it is not described how they were constructed. Please add this information to the figure legend and/or methods section. I can't understand this "Color key and Histogram". What is "value" and "count"? The "count" axis goes from 0-80000, but I don't see anything in the plot, I guess the histogram is missing in this plot?
A: We thank the reviewer for this constructive feedback and agree that the main findings were not well presented in the heatmap. We have therefore revised the heatmap, so that patterns of differentially abundant features across left and right armpits and different putative chemical classes become more apparent. We have reduced the heatmap so that it only contains data from left and right armpits as suggested by the reviewer. Furthermore, we limited the amount of metabolite features displayed to only features, which showed significantly differentially abundant (Kruskal-Wallis, FDR-adjusted P < 0.05) across left and right armpits:

Supplementary Figure 4
Heatmap of metabolites from armpits that have different distributions within left and right armpit samples of all volunteers combined at all time points. All features displayed are significantly differentially abundant (Kruskal-Wallis, FDR-adjusted P < 0.05) across left and right armpits. Chemical classes are putative estimates retrieved through the MolNetEnhancer 58 workflow and based on similarities in MS2 fragmentation patterns. Relative metabolite abundances are scaled for better pattern visualization.
We believe that chemical differences across different putative classes are now clearly visualized, and we further clarified the sentence in the main manuscript on page 7 highlighted by the reviewer to: "A number of compound classes that may be linked to compounds of bacterial origin -such as acylcarnitines (organonitrogen compounds), glycerophospholipids and bile acids (steroids and steroid derivatives) have been found to have different abundances in the two armpits (Supplementary Figure 4)." In addition, we added a detailed description to the material and methods section, where we describe how the heatmap was created as well as how rows and columns were clustered (euclidean distance and complete clustering method): A Kruskal-Wallis test was used to find differentially abundant metabolites across left and right armpits and P values were adjusted for multiple hypothesis testing using the false discovery rate (FDR) method [ref]. To visualise different distributions of metabolites across left and right armpits a heatmap of the differentially abundant metabolites (FDR-adjusted P < 0.05) was created using the ComplexHeatmap package version 2.8.0 [ref] in R. Rows and columns were clustered using the euclidean distance and complete clustering method. Only differentially abundant metabolites with a putative class annotation were displayed in the heatmap. The Jupyter notebook used to create the heatmap can be found at https://github.com/.
To clarify how putative metabolite annotations were retrieved, we have furthermore added an additional paragraph in the Methods section called "Metabolite annotation":

Metabolite annotation
MZmine preprocessed MS/MS fragmentation spectra were submitted to feature-based mass spectral molecular networking through the Global Natural Products Social Molecular Networking Platform (GNPS) [refs] and searched against all GNPS spectral libraries. To further enhance chemical structural information we performed in silico structure annotation using Network Annotation Propagation [ref] and created consensus chemical classes per molecular family using the GNPS MolNetEnhancer workflow (https://ccmsucsd.github.io/GNPSDocumentation/ molnetenhancer/)   This is a very interesting work that brings some information about the composition of the skin microbiota under the effect of fabrics containing silver with antimicrobial claims. The experimental design was well executed and the results were quite promising, but I had some questions about the discussions.
Regarding chemical diversity, it was not clear to me how this result was obtained, I understood that the Shannon diversity index was used, but exactly how was the index calculated? For example, looking at figure 1 a) I would expect a greater number of only green nodes, showing that there is a greater number of molecules without the presence of silver. But what I see seems to be the opposite, the difference is small, but a greater amount of only grey nodes are observed, which would indicate a greater production of different molecules in the presence of silver, correct?
The other observations I found all very interesting and consistent with the data. Just out of curiosity, did you investigate other influences such as climate, which could be equally affecting all the volunteers in the different periods (with silver and without silver clothes). Because as the two periods (groups) were run at different times, maybe the results could be masked by a variable that affects everyone equally. A: We have added this crucial information in the Study Design section of M&M: "Participants all lived in the temperate climate and region of San Diego during the time of study (April-June 2017)." Did you collect sensory information from the participants? If they noticed a difference in odours, skin appearance, etc. A: Very good question -we have not performed odour assessments on the shirts or participants, as it was not part of the study design. We have noted down remarks and feedback participants provided, such as for example acne formation or skin feel. These provided us with meaningful insights.

Did you do any evaluation of volatile compounds?
A: We have performed LC/MS, accounting for mainly the non-volatile compounds. Volatile compounds were also captured with the sampling, but were incomplete and unreliable at this point to draw meaningful conclusions. In a follow-up study, this would definitely be a meaningful parameter to take into account. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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